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Prevalence of potentially pathogenic enteric organisms in clinically 6

healthy kittens in the UK. 7

8 Adam G Gow

BVM&S CertSAM MRCVS

1*, Deborah J Gow BVM&S MRCVS

2, Edward J. 9

Hall MA VetMB PhD DipECVIM-CA MRCVS3, Deborah Langton

4, Chris Clarke HNC

5, 10

Kostas Papasouliotis DVM PhD DipECVCP DipRCPath MRCVS5

11 12 1Hospital for Small Animals, Division of Veterinary Clinical Studies, R(D)SVS, University of Edinburgh, Easter 13 Bush, Midlothian EH25 9RG 14 15 2Division of Companion Animal Sciences, University of Glasgow Faculty of Veterinary Medicine, Glasgow, G61 16 1QH 17 18 3 Division of Companion Animal Studies, Department of Clinical Veterinary Science, University of Bristol, Bristol 19 , BS40 5DU 20 21 4Division of Veterinary Pathology, Infection and Immunity, Department of Clinical Veterinary Science, 22 University of Bristol, Bristol, BS40 5DU 23 24 5Langford Veterinary Diagnostics, Department of Clinical Veterinary Science, University of Bristol, Bristol, BS40 25 5DU 26 27

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*Corresponding author email: adam.gow@ed.ac.uk 40

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Summary 41

Faecal samples were collected from 57 clinically healthy kittens presented for initial 42

vaccination, in the UK. Routine bacteriological examination identified Salmonella in 43

1 and Campylobacter in 5 samples. PCR detected the presence of Campylobacter in 44

further 4 samples. Routine parasitological examination revealed Toxocara ova in 9 45

(including 4 kittens stated to have been administered an anthelmintic) and Isospora in 46

4 samples. No Giardia or Cryptosporidium were detected by routine methods. A 47

Giardia ELISA test kit designed for use in cats was positive in 3 kittens. A similar 48

test kit designed for use in humans was negative in all samples and produced negative 49

results even when positive control samples were tested. Potentially pathogenic enteric 50

organisms were detected in 19 kittens by routine methods and 26 (prevalence 45%) by 51

all methods. The high prevalence in asymptomatic kittens highlights the possibility 52

that the detection of these organisms in kittens with gastro-intestinal disease may be 53

an incidental finding. 54

55

Introduction 56

Campylobacter, Salmonella, protozoa, and helminths are frequently implicated as the 57

cause of gastrointestinal disease when they are isolated from cats exhibiting 58

compatible clinical signs. The true significance of these organisms in causation of 59

clinical signs is unclear as the prevalence of these organisms in asymptomatic animals 60

in the UK has not been investigated. In a study of cats less than 1 year old which 61

were living in New York (Spain and others, 2001) the presence of diarrhoea was not a 62

reliable indicator for the presence of potentially zoonotic enteric organisms. Thus it 63

is possible that detection of these organisms in symptomatic cats may be an incidental 64

finding. 65

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66

Certain of these enteric organisms also have the potential to cause disease in humans 67

and their presence in asymptomatic cats may be a source of infection for their owners. 68

Although studies have shown that cat ownership is an increased risk factor for the 69

development of some diseases, such as campylobacteriosis (Deming et al 1987, Saeed 70

et al 1993), others have reported this to be an insignificant variable when investigating 71

the incidence of these diseases in humans (Cook et al 2000, Potter et al 2003, Tenkate 72

and Stafford 2001). In addition, Heyworth and others (2006) demonstrated that dog 73

or cat ownership appeared to be protective against the incidence of gastroenteritis in 74

children. Thus the role of companion animals in transmission of these organisms to 75

humans is unclear. Veterinary surgeons and human doctors may be asked for advice 76

regarding the risk of owning a cat, especially if a member of the household is 77

immunocompromised. Prevalence of these enteric organisms in clinically healthy pet 78

cats would provide more information on the potential for zoonotic spread and assist 79

veterinary surgeons and doctors in counselling at-risk owners. 80

The aim of this study was to sample clinically healthy kittens presented to veterinary 81

surgeons in the UK in order to identify: 1) the prevalence of Campylobacter spp., 82

Salmonella spp. and enteric parasites using standard laboratory methods, 2) the 83

prevalence of Campylobacter spp. and Salmonella spp. using polymerase chain 84

reaction (PCR) techniques, and 3) the prevalence of Giardia spp using two 85

commercially available rapid immunoassays; one designed for cats and dogs (SNAP 86

Giardia, IDEXX Laboratories, Westbrook, USA) and one for humans (Giardia-Strip, 87

CORIS BIOCONCEPT, Gembloux, Belgium). Information on signalment, source of 88

the kitten, anthelmintic history and presence of gastrointestinal disease was also 89

sought. 90

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91

Methods 92

Sampling 93

Between May 2006 and June 2007, 14 first opinion veterinary practices in mainland 94

UK distributed faecal sampling kits consisting of 3 sample pots, disposable gloves 95

and a questionnaire to owners who presented their kittens (age range 9-20 weeks) for 96

the initial vaccination course. 97

The owners were instructed to collect one faecal sample each day for 3 days from 98

their kitten and complete the questionnaire. The questionnaire (Figure 1) requested 99

details on signalment, where the owner acquired the kitten, previous anthelmintic 100

treatment (including preparation if known) and faecal consistency. The samples and 101

completed questionnaires were then posted to Langford Veterinary Diagnostics at the 102

University of Bristol. 103

On arrival, standard laboratory methods were employed on pooled faecal samples for 104

bacteriological culture and parasitological examination, while the remaining samples 105

were stored at -20º C. The questionnaires and laboratory test results were faxed to the 106

Hospital for Small Animals, Royal (Dick) School of Veterinary Studies for analysis 107

108

A follow-up questionnaire, two to nine months after the submission of the initial 109

questionnaire, was completed and submitted by the owners. This questionnaire was 110

identical to the first one with the addition of one question, enquiring whether the 111

kitten had required veterinary attention for gastro-intestinal problems. 112

113

114

Bacteriological cultures 115

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Campylobacter 116

Pooled faeces were inoculated onto plates with Campylobacter Blood-Free Selective 117

agar plates (Oxoid CM0739B, Oxoid Ltd, Basingstoke, UK) with CCDA selective 118

supplement (Oxoid SR0155E). Plates were then incubated at 37ºC in a 2.5 L jar 119

containing “Campygen” (Oxoid CD0025A) for 3 days. Colonies which produced a 120

positive Oxidase test and revealed curved/”seagull” shaped negative rods under 121

microscopy after Gram staining, were reported as Campylobacter spp. 122

Salmonella 123

Pooled faeces were inoculated onto MacConkey agar (Oxoid CM007B), DCLS agar 124

(Oxoid CM0393B) plates and into 20ml universal containers with Selenite enrichment 125

broth (Oxoid CM0395B+LP0121). Plates and broth were incubated at 37ºC under 126

normal atmospheric conditions overnight. After incubation, the Selenite broth was 127

sub-cultured to a DCLS agar plate which was incubated overnight. Non-lactose 128

fermenting colonies were picked off into urea broth and incubated at 37ºC for 4 hours. 129

Urea negative cultures were sub-cultured to triple sugar iron (TSI, Oxoid CM0277) 130

slopes and incubated at 37ºC overnight. TSI slopes generating positive reactions for 131

Salmonella spp were sub-cultured to AP120E identification strips (BioMerieux 132

20100, BioMerieux, Marcy, France) for confirmation. Agglutination testing using 133

polyvalent and monovalent Salmonella anti-sera was also carried out. 134

135

PCR techniques 136

Preparation of DNA extracts 137

Faecal samples were defrosted and DNA was extracted using the QIAamp DNA Stool 138

mini kit (Qiagen Ltd., Crawley, UK) following the manufacturer’s instructions. PCR 139

was performed in a DNA Engine (MJ Research, Inc., Waltham, MA, USA). All PCR 140

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amplicons were electrophoresed on a 1.5 % agarose gel containing 1 µg ml-1

ethidium 141

bromide (Sigma-Aldrich, Dorest,UK), and visualised on an ultra violet 142

transilluminator (UVP BioDoc It imaging system, Cambridge, UK). 143

144

Triplex PCR 145

A novel triplex polymerase chain reacton (PCR) assay, developed in-house using 146

previously described primers (Aabo et al 1993, Bej et al 1991, Croci et al 2004, 147

Linton et al 1996, Steinhauserova et al 2000) was used to enable the simultaneous 148

detection of, Salmonella spp. and Campylobacter spp. (Table 1). 149

150

Multiplex PCR 151

A modification of the method described by Wang and others (2002) was used for 152

Campylobacter speciation (Multiplex PCR). Each PCR reaction contained 1 x hotstar 153

taq (Qiagen), 1.5 mM MgCl2, 0.3 µM of each C. jejuni primer, 0.6 µM of each of 154

the C. coli, C. lari and C. fetus primers, 0.13 M of each 23S rDNA primer, 1.25 µM 155

of each C. upsaliensis primer and 1 µl of whole-cell template DNA. This multiplex 156

was used to speciate Campylobacter detected either with Triplex PCR or routine 157

bacteriological culture. 158

The PCR assay was modified further to improve the speciation of C. jejuni and C. coli 159

by combining only C. jejuni and C. coli primers (Duplex PCR). Each PCR reaction 160

contained 1 x hotstar taq (Qiagen), 1.5 mM MgCl2, 0.65 µM of each C. jejuni 161

primer, 1.3 µM of each C. coli primer, 0.13 µM of each 23S rDNA primer and one µl 162

of whole-cell template DNA. This duplex assay was performed only on 163

Campylobacter culture positive samples. 164

165

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Parasitological examination 166

Following centrifugation of faecal samples in a zinc sulphate solution (specific gravity 167

1.18), a sample of the surface fluid was removed via a 10l loop or plastic pipette and 168

positioned on a glass slide which was then examined using a x40 objective for the 169

microscopic identification of parasitic ova, oocysts and cysts. Thin, air-dried faecal 170

smears were also prepared, stained with a modified Ziehl-Neelsen stain and examined 171

microscopically using a x100 oil objective for the identification of oocysts with 172

morphological characteristics typical of Cryptosporidium spp. 173

174

Rapid immunoassays 175

Faecal samples were defrosted in batches of five and both immunoassays for the 176

detection of Giardia antigen (SNAP and Strip) were performed according to the 177

manufacturers’ instructions. A faecal sample positive for Giardia oocysts on 178

microscopy was also included and tested with each batch and acted as an internal 179

positive control. 180

Statistical analysis 181

Questionnaire and laboratory data was entered into a spreadsheet (Microsoft Excel, 182

Washington USA). A statistical package (GraphPad Prism version 5.01 for Windows, 183

GraphPad Software, San Diego USA) was used to calculate prevalence rates with 184

95% confidence intervals and χ2 test for significant associations. Significance was set 185

at p<0.05. 186

187

Results 188

Questionnaires 189

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Fifty-seven sets of faecal samples and completed questionnaires were returned from 190

14 veterinary practices (Figure 2). The median age was 11.6 weeks (range: 9-20 191

weeks). Fifty-four percent of the kittens were male. Domestic short and long-haired 192

cats made up 81% of the population. Siamese and Burmese each represented 5%. 193

Eighteen percent of kittens had been acquired from a rescue centre or charity, 61% 194

from an owner whose cat had kittens and 14% from a breeder (all pedigree cats). The 195

remaining kittens had been acquired from a pet shop (3 kittens) or bred from the 196

owner’s cat (1). Anthelmintic medication was stated to have been administered in 197

77% of kittens. Fenbendazole was the most frequently stated anthelmintic (25%), 198

although 43% of owners could not recall the name or type of the wormer used. 199

200

Bacteriological culture 201

Bacterial cultures were performed on 57 samples. 202

Salmonella spp was detected in 1 kitten. The follow-up questionnaire for this kitten 7 203

months later reported absence of any clinical signs and no need for veterinary 204

attention. 205

Campylobacter was cultured from 5 kittens (8.7%, 95%CI 3.4-19.3%). 206

207

PCR assays 208

Triplex PCR testing was performed in 54 samples. 209

All samples were negative for Salmonella spp., including the culture positive sample. 210

Campylobacter spp. was detected in 4 samples, but not in any of the 5 culture positive 211

samples. Speciation by Multiplex PCR identified two of the 4 Campylobacter species 212

as C. jejuni. 213

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Of the 5 Campylobacter spp culture positive samples, the Multiplex PCR was 214

negative in 4 and positive (C. upsaliensis) in one sample. The Duplex PCR was 215

negative for all 5 culture positive samples. 216

217

Parasitological examination. 218

Routine parasitological examination was performed on 57 samples. All samples were 219

negative for Giardia cysts and Cryptosporidium oocysts. Isospora felis cysts were 220

present in 4 samples (7%, 95%CI 2.3-17.2%). Three of these kittens were reported to 221

have soft faeces between once to 50% of the time. From the follow-up questionnaire 222

2-8 months later, none of these kittens had required veterinary attention for gastro-223

intestinal disease and faecal consistency was reported to be normal. 224

Toxocara ova were identified in 9 kittens (15.7%, 95%CI 8.3-27.5%). The 225

questionnaires revealed that an anthelmintic had been administered in five of these 226

animals. The difference in prevalence of Toxocara between those kittens stated to 227

have an anthelmintic administered (9%) and those not (33%), was not statistically 228

significant (p= 0.082). 229

230

Rapid immunoassays 231

SNAP and Strip tests for the detection of Giardia antigen were performed in 55 232

samples. The SNAP test was positive in 3 samples and always generated a positive 233

result when the internal positive control sample was tested. The Strip test was 234

negative on all 55 samples but also generated a negative result every time the internal 235

positive control sample was analysed. 236

237

Prevalence of enteropathogenic organisms 238

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Campylobacter spp., Salmonella spp. and/or enteric parasites were detected in 19 239

kittens by standard laboratory methods, a prevalence of 33% (95%CI 22.5-46.3%). 240

Using PCR assays, only Campylobacter spp. was detected in 4 kittens, a prevalence of 241

7.4% (95% CI 2.4-18%). The prevalence of Giardia using the SNAP test was 7.2% 242

(95% CI 2.4-17.7%). Combining the results of all techniques, organisms were 243

detected in 26 animals, a prevalence of 45%. (95% CI 33-58%). No animal had 244

required veterinary attention for gastrointestinal disease at follow-up. No kitten had 245

more than one organism of pathogenic potential isolated. 246

247

Discussion 248

The population of cats employed in the present study was selected as the most likely 249

to cause transmission of enteric organisms to humans; kittens may not be fully house-250

trained and can cause faecal contamination in the house. It was thought that kittens 251

would be most likely to use a litter tray and transmission may occur during emptying 252

of these. In addition, a number of studies have shown that young cats have a higher 253

prevalence of enteric organisms of zoonotic potential than older cats (Acke et al 2006, 254

De Santis-Kerr et al 2006, McGlade et al 2003, Shukla et al 2006, Tzannes et al 255

2008). 256

257

The prevalence of Toxocara in the present study (15.7%) is lower than the one 258

reported (27.2%) by Spain and others (2001) in healthy client-owned less than 1 year 259

old cats. However the proportion of cats that received anthelmintic treatment was not 260

reported in that study. Although in our study there was no significant difference in 261

the prevalence of Toxocara between kittens that were administered an anthelmintic 262

and those that were not, it is likely that a larger sample size would have demonstrated 263

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a statistically significant difference. The presence of Toxocara in animals which 264

were stated to have had an anthelmintic is a potential concern. Patent infection can 265

occur with infrequent worming or administration of an incorrect dose. In the current 266

study, the questionnaire did not elicit the timing of administration and it is possible 267

that administration of the anthelmintic may have been after or during sample 268

collection, and the animal would have been subsequently negative for Toxocara. The 269

presence of Toxocara underlines the need for continued client education and regular 270

anthelmintic treatment in this population. 271

No samples were found to contain Cryptosporidium oocysts. Tzannes and others 272

(2007) detected a prevalence of only 1% in a UK study employing 1355 cats, most 273

with gastrointestinal signs. Although Cryptosporidium may be absent from the 274

present population due to an association with gastrointestinal disease, it is possible 275

that the current study population may contain similar prevalence but due to the low 276

numbers in the present study this would not have been detected. To have a 95% 277

confidence of detecting one positive sample using a population prevalence of 1% with 278

a 100% sensitive test, a sample size of 299 would have been required. Hill and others 279

(2000) after testing 205 cats (<1 to > 10 years old), with and without diarrhoea stated 280

that Cryptosporidium was the most prevalent zoonotic agent detected (5.4%). The 281

majority of positive results were generated by employing an ELISA test rather than 282

the ZN stain. Utilising the ELISA test may have resulted in the detection of the 283

organism in our sample population 284

285

The 8.7% prevalence of Campylobacter by culture in the current study is similar to 286

the prevalence of 5%, reported by Hald and Madsen (1997) who sampled 42 healthy 287

kittens aged between 11-17 weeks, but much lower than the 41.9% prevalence in the 288

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study of healthy cats reported by Wieland and others (2005), In both studies, stricter 289

sample handling was undertaken with culture being performed within 48 hours of 290

sampling. Routine culture for Campylobacter by Bender and others (2005), using 291

samples from both ill and healthy cats which were posted to the laboratory in a similar 292

method to the current study detected a prevalence of 24%. A study comparing 293

sampling handling and culture techniques would be required to investigate if these 294

apparent differences in prevalence are due to true population difference or study 295

methodology. Hill and others (2000) detected a prevalence of 1.9% in 52 healthy 296

client owned cats although only C. jejuni was investigated. 297

298

The prevalence of Salmonella (1/57) in this study is of a similar magnitude to the one 299

reported by Van Immerseel and others (2004) where out of 278 healthy Belgian house 300

cats only one found to be excreting the organism. Similar prevalence was also 301

reported by Hill and others (2000) where all faecal samples from 52 healthy client-302

owned cats were negative for Salmonella by culture. 303

Triplex PCR detected the presence of Campylobacter in 4 kittens but did not detect 304

the organism in any of the five samples with positive cultures. The discrepancy 305

between Campylobacter culture and PCR results could suggest that sensitivity of each 306

may have caused underestimation of prevalence. This discrepancy may be due to 307

multiple factors. PCR will detect non-viable bacteria with intact DNA sequences, 308

allowing detection in culture negative samples. A positive PCR result depends on 309

sufficient quantity and quality of the sequence under investigation as well as lack of 310

substances which may interfere with amplification. Freezing and storage of samples 311

prior to PCR analysis may have caused disruption of bacterial DNA. Batching of the 312

three samples may have reduced the quantity of DNA to below detection threshold if 313

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the organism was only excreted intermittently. These factors may explain the samples 314

which were culture positive but PCR negative. PCR testing of each sample separately 315

at the time of arrival at the laboratory would have avoided these factors. PCR 316

sensitivity may be inherently less than culture in viable samples; faecal material 317

contains PCR inhibitory components which reduces sensitivity (Radstrom et al 2004). 318

319

Speciation by Multiplex PCR failed to identify the type of Campylobacter in 4 of the 320

5 culture positive samples and in 2 of the 4 Triplex PCR positive samples. This may 321

have been due to inherent PCR problems as detailed above or the presence of a 322

species not included in the primer set. For example, Weiland and others (2005) found 323

a very high prevalence of C. helveticus amongst their population of healthy cats. 324

Primers for this species were not present in the Multiplex PCR. 325

326

Giardia was detected only using the SNAP immunoassay. Mekaru and others (2007) 327

found that this test had an identical sensitivity to flotation when compared to direct 328

immunofluorescence as the gold standard. Although no gold standard test was 329

performed in the current study, Mekaru and others (2007) found this test to be 100% 330

specific, therefore it is likely that these are true positives. The prevalence in the 331

current study (5.4%) is very similar to the prevalence of 6.1% found by Vasiloupulos 332

and others (2006) in healthy cats when direct immunofluorescent antibodies were 333

used. Tzannes and others (2008) also found a similar prevalence in healthy UK cats 334

using another commercial ELISA (4%). The human Giardia immunoassay failed to 335

detect Giardia in any samples or the positive control samples. This human 336

immunoassay kit has been reported to have a relatively low sensitivity; 58% (Oster et 337

al 2006) and 44% compared with 88-80% sensitivity of the other kits tested (Weitzel 338

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et al 2006). It is possible that the lack of detection in these feline samples is partly a 339

reflection of this low sensitivity. Giardia immunoassays designed for human samples 340

also performed poorly in the Mekaru and others (2007) study. Mekaru and others 341

(2007) postulated that genetic differences between common animal/human Giardia 342

isolates may explain the poor performance of the human assay. There is evidence that 343

the more common genotypic isolate from cats (i.e. assemblage type F) is not involved 344

in human infection, although the zoonotic assemblage type A has been identified in 345

cats (Vasilopulos and others, 2007). The sensitivity to different Giardia assemblages 346

of the two kits used in the current study is unknown. Mekaru and others (2007) 347

concluded that caution should be exercised when using human-based immunoassays 348

for parasite detection in animals. This current study supports this warning. 349

350

This study is likely to have underestimated the prevalence of these organisms for a 351

number of reasons. Excretion of many of these organisms is intermittent (Barr 2006, 352

Fox 2006). Although three samples were submitted, these were collected within a 353

short time period and episodes of excretion may have been missed. The sampling 354

method may not have been optimal. Acke and others (2006) found a higher rate of 355

detection of Campylobacter in cats where collection was by rectal swab rather than 356

faecal sample. It is unknown if this difference was due to sampling method or 357

increased prevalence in the animals swabbed. Toxocara may have been present but 358

not excreting eggs at the time of sampling or not detected by flotation (Lillis 1967, 359

Wolfe et al 2001). The delay and transport conditions between sample collection and 360

receipt at the laboratory may have reduced the viability of Campylobacter for culture 361

(Greene 2006) and/or disrupted protozoal organisms, making identification impossible 362

(Barr 2006). Even so, the protocol for sampling and transport mirrors sampling in 363

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first opinion veterinary practices that submit samples to external laboratories and 364

therefore the generated prevalence could be compared with the prevalence in 365

symptomatic animals. 366

367

The use of PCR and ELISA techniques more than doubled the apparent detection rate 368

of potentially enteropathogenic organisms. With any diagnostic laboratory test, there 369

is the possibility of false-positive results. Although regular quality control 370

assessments are performed at the laboratory and both positive and negative controls 371

were used during PCR testing, this possibility cannot be ruled-out, especially as no 372

“gold-standard” test was available to evaluate further the disparate results between 373

routine laboratory tests and the more advance techniques. 374

375

The high prevalence of these enteric organisms in this ostensibly healthy population 376

raises a number of issues. These kittens were presented for initial vaccination and 377

not for the presence of gastrointestinal disease. If these organisms were detected in 378

animals with gastrointestinal disease they would likely be implicated as the cause. It is 379

possible that when detected in diseased animals they may be incidental findings or if 380

these organisms are causing gastrointestinal disease, there may be concurrent factors 381

which allow this. Ultimately, a case-control study utilising identical sample handing 382

and laboratory methods would be required to attempt to identify any difference in 383

prevalence of these organisms between animals with gastro-intestinal disease and 384

clinically normal animals. It is still possible that perturbations in the gastro-intestinal 385

environment (e.g. change in osmolarity or transit time) due to unrelated gastro-386

intestinal disease may allow overgrowth and therefore increased detection rates of 387

organisms. 388

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389

It could be argued that the high prevalence of potential zoonotic organisms suggests 390

that cats may be a significant source of infection for humans. The mere detection of 391

these organisms, although indicating prevalence in this population, does not 392

necessarily imply a zoonotic risk. Many of these organisms vary in pathogenicity to 393

humans depending on their genotype (Fayer et al 2004, Fox 2006, Palmer et al 2008, 394

Vasilopulos et al 2007). Thus further typing of these organisms would be required to 395

assess their true zoonotic potential. The number and viability of organisms excreted 396

is an important factor in environmental contamination and subsequent zoonotic risk. 397

PCR techniques are designed to identify presence of organisms at very low levels. A 398

method of organism quantification may be useful in assessing risk, such as 399

quantitative real-time PCR. 400

401

The converse argument to zoonotic risk is that if these organisms are this prevalent, it 402

would be expected that if cats are a significant source of human infection, then more 403

human epidemiological studies investigating risk factors for these diseases would 404

have identified contact with cats as a risk. This has not been the case with more 405

studies showing pet owning as having a protective affect against the development of 406

gastroenteritis and/or infection with these organisms (Heyworth and others 2006, 407

Robertson et al 2002) or cats having no association with development of disease in 408

humans (Cook and others 2000, Glaser et al 1998, Potter and others 2003, Tenkate 409

and Stafford 2001) than contact with cats increasing the risk of these diseases 410

(Deming and others 1987, Saeed and others 1993). Thus it appears that although 411

prevalence of these organisms is high in this population of cats, this does not 412

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necessarily translate into either clinical signs of disease or zoonotic transmission to 413

humans. 414

415

Acknowledgements 416

The author’s would like to thank the Clinical Research Outreach Programme (CROP) 417

for providing funding for this study. A.G. would like to thank Hill’s Pet Nutrition for 418

providing funding for his residency. Idexx is gratefully acknowledged for providing 419

their test kits at reduced cost. 420

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